Project Summary/Abstract Sepsis is a complex lethal condition defined as ?life- threatening organ dysfunction caused by a dysregulated host response to infection?. In the U.S, sepsis is related to ~1 million hospitalizations annually, and the incidence of sepsis continues to rise. The mortality rate from in patients with sepsis remains unacceptably high. Novel data from our laboratory recently demonstrated that simply moving the spleen to midline and then returning it to its original position causes development of splenic capsular fibrosis and a pro-inflammatory shift in the immune cell phenotype of the spleen. While similar findings have been reported in mice, it remains unclear how these maneuvers promote a pro-inflammatory phenotype. Our novel data indicate that specialized mesothelial cells that form connections to the splenic capsule may transmit tonic anti-inflammatory signals to spleen via cholinergic signaling through alpha 7 nicotinic receptors. In addition to markers of cholinergic signaling, these mesothelial cells strongly express neuronal markers and display neuronal-like organelles within their cytoplasm. As the connections themselves are made of collagen lined with mesothelial cells, splenic capsular fibrosis following disruption of these connections may represent a physiological response to try to re-establish mesothelial cell to cell signaling. The primary goal of this proposal is to determine whether cholinergic signaling through specialized mesothelial cells does indeed transmit tonic anti-inflammatory signals to the splenic capsule and whether disruption of this signaling has a functional response, exacerbating the systemic inflammatory response to endotoxin. As such, Aim 1 will test whether `Surgical disruption of mesothelial signaling to the splenic capsule results in a prolonged anti-inflammatory profile and exacerbated inflammatory response to endotoxin'. We will access the spleen in rats via a flank incision and clear the spleen of mesothelial connections without interfering with the spleen. Baseline immune profiles (flow cytometry/cytokine analysis) and the physiological response to experimental inflammatory shock (endotoxin) in anesthetized disrupted and sham surgical rats will be determined 24 hours, 21 days and 3 months following surgical disruption of mesothelial connections to the spleen Aim 2 will test whether `Non-neuronal cholinergic signaling between mesothelial cells mediates a tonic anti-inflammatory response in the spleen'. Global deletion of ?7nAChR in mice results in a pro-inflammatory splenic phenotype however, the cell type(s) mediating this effect remain unclear. We will use Wilms tumor 1 homolog gene promoter Cre and Chrna7tm1 floxed mice to `knockout' ?7nAChR specifically in mesothelial cells. We predict that loss of the ?7nAChR specifically in mesothelial cells will promote a pro-inflammatory phenotype in the spleen. If our hypothesis is correct, neuronal like signaling between distal sites through mesothelial cells would represent a previously unrecognized pathway of biological communication and would likely have a major impact not only on our understanding of the factors that regulate systemic immune responses in sepsis, but also idiopathic fibrosis of other organs involving the mesothelium.